Fibrinolysis

ABSTRACT

A method for degrading fibrin deposits and preventing such deposits associated with pathological conditions is described, which comprises administering to a subject in need of such treatment a therapeutically effective amount of IL-4 activity, optionally in association with one or more pharmaceutically acceptable carriers or excipients. There is also described thrombolytic compositions which comprise IL-4 or a derivative thereof possessing IL-4 activity together with a pharmaceutically acceptable carrier or excipient.

The present invention relates to fibrin degradation or breakdown (whichmay be referred to as fibrinolysis), and compositions and methods forthe treatment of pathological conditions associated with fibrindeposition.

Fibrin plays a crucial role in haemostasis and wound healing, and islaid down in the human and animal body as a result of a complex seriesof biochemical reactions. Notwithstanding these crucial functions offibrin, fibrin formation is also a common event in many pathologicalconditions and inflammatory lesions. For example, fibrin deposition isassociated with atherosclerosis, rheumatoid arthritis,glomerulonephritis, systemic lupus erythematosis, myocardial infarcts,pulmonary embolism, deep vein thrombosis, autoimmune neuropathies,granulomatous disease, parasitic infections and allograft rejection.Metastases, or so called "secondary" tumours, have been linked withthromboembolism phenomona. The abundant fibrin deposited around somesolid tumours in the stroma may serve as a cocoon that hinderslymphocytes, macrophages and other inflammatory cells from reachingtumours.

Fibrin deposition may also be associated with renal disease andhypertrophic scars and keloids. Fibrous adhesions are also a significantproblem in post-operative surgery.

Urokinase and tPA (tissue plasminogen activator) are plasminogenactivators (PA's) which have previously been used both experimentallyand clinically to effect fibrin lysis, and in particular, the lysis ordegradation of fibrin clots. Whilst effective in degrading fibrin, thesemolecules have attendant disadvantages. Urokinase activates plasminogento give plasmin independently of the presence of fibrin (unlike tPA),and thus large amounts must be administered to effect fibrinolysis, thisbeing expensive and causing unwanted bleeding. tPA has a short half lifein vivo, and thus large quantities have to be administered over a longperiod of time, resulting in unwanted bleeding and expense.

It has now surprisingly been found that the lymphokine, interleukin-4(IL-4), activates both human and animal cells to produce plasminogenactivators, which results in fibrin degradation. Also, IL-4 inhibits theprocoagulant activity of human monocytes resulting in decreased fibrinformation at the monocyte surface.

IL-4 is a lymphokine, which exhibits both B cell and T cell growthfactor activities (published Australian Patent Application No.67334/87). This lymphokine also exhibits suppressive activity, as itsupresses human monocyte production of the cytokines lL-1, TNF, andsuppression of PGE₂.

IL-4 has been purified to homogeneity, and the gene encoding thisprotein has been cloned allowing IL-4 to be produced in large amounts byrecombinant DNA technology, as described in published Australian PatentApplication No. 67334/87, in the name Schering-Biotech Corporation. IL-4(human) is commercially available from a number of suppliers.

On the basis of IL-4's activity in suppressing cytokine production, itwas most surprising that IL-4 was effective in stimulating plasminogenactivator (PA) production, notably, t-PA and urokinase, by appropriatetarget cells.

In accordance with the present invention, there is provided a method fordegrading fibrin deposits and preventing such deposits associated withpathological conditions or which may lead to such conditions, whichcomprises administering to a subject in need of such treatment atherapeutically effective amount of IL-4 or a derivative thereofpossessing IL-4 activity, optionally in association with one or morepharmaceutically acceptable carriers or excipients.

Pathological conditions which may be treated in accordance with theinvention are those which are caused wholly or at least in part byfibrin deposition. These include deep vein thrombosis, pulmonaryembolism, renal disease, hypertrophic keloid scars, coronary infarction,metastasis, inflammation, disseminated intravascular coagulation,atherosclerosis, rheumatoid arthritis, glomerulonephritis, systematiclupus eryttematosis, autoimmune neuropathies, granulomatous disease,parasitic infection, allograft rejection, and other conditionsassociated with fibrin deposition. Administering IL-4 to subjectssuffering from such conditions may result in increased plasminogenactivator, particularly tPA and urokinase (prourokinase) production bytarget cells, causing activation of plasminogen and subsequent fibrindegradation. Also, fibrin formation in such patients may be lessened.

Notable cell types stimulated to produce t-PA when stimulated with IL-4are the monocyte/macrophage cell types. Importantly, these cell typesare often closely associated with thrombi and other fibrin deposits, andhence, when stimulated to produce plasminogen activators on contact withIL-4, may cause highly efficient localised fibrin degradation. Thisavoids the need to administer large amounts of plasminogen activator toa patient, hence reducing side effects of unwanted bleeding, and excesscost due to the administration of large amounts of plasminogenactivator.

Endothelial cells may also be stimulated with IL-4 to produce urokinase(prourokinase). This is also of importance as fibrin deposits aregenerally associated with endothelial cells.

Other target cells within a human or animal subject may also bestimulated with IL-4 to effect fibrin degradation. A number of othertarget cells besides the monocyte may also loose their ability toproduce procoagulant activity as a result of IL-4 action.

Preferably, the IL-4 employed in this invention is produced byrecombinant DNA technology. Where human subjects are to be treated, itis of course desirable that the IL-4 is of human origin. Where animalsare treated, animal IL-4 is preferred.

Any IL-4 derivative or analogue which possesses the B cell, T cell andmast cell stimulatory effects of IL-4, and other characteristicactivities of IL-4, as described in published Australian PatentApplication No. 67334/87 may be used in this invention. The precisenature of IL-4 derivatives or analogues is not of importance. Rather,the derivatives or analogues must possess the well defined biologicalactivity of IL-4, and, must possess the ability to stimulate targetcells such as macrophages/monocytes to produce plasminogen activators.Application No. 67334/87 mentioned above teaches methods for theproduction of IL-4 derivatives and analogues. As used in thisspecification the term IL-4 encompasses human IL-4, IL-4 of animalorigin such as murine, ovine or bovine IL-4, and analogues orderivatives thereof which possess characteristic IL-4 activity. Forconvenience these molecules may be referred to hereinafter as "activematerial".

IL-4 may be administered in a convenient manner such as by the oral,intraveneous, intramuscular, subcutaneous, intraperitoneal, intranasal,intradermal or suppository routes.

IL-4 may also be administered to a human or animal subject by continuousinfusion over a predetermined time period, for example, from 30 minutesto 24 hours. Administration may be by way of an intravenous catheterconnected to an appropriate pump, or by gravity feed.

The amounts of and dosage regimes of IL-4 which are administered to asubject to effect fibrin degradation will depend on a number of factorssuch as the mode of administration, the nature of the condition beingtreated, the body weight of the subject being treated, and the judgementof the prescribing physician or veterinarian. Generally speaking, IL-4may be administered in an amount between 0.1 μg to 2000 mg per kilogramof body weight per day. The quantity of active compound in a unit dosagesuch as a tablet or capsule may vary from about 0.1 μg to 100 mg.

IL-4 may be coated by, or administered with, a material to prevent itsinactivation. For example, the active material may be administered in anadjuvant, co-administered with enzyme inhibitors or in liposomes.Adjuvants contemplated herein include resorcinols, non-ionic surfactantssuch as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether.Enzyme inhibitors include pancreatic trypsin inhibitor,diisopropylfluorophosphate (DFP) and trasylol. Liposymes includewater-in-oil-in-water P40 emulsions as well as conventional liposomes.

Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof and in oils. Under ordinary conditions ofstorage and use, these preparations contain a preservative to preventthe growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases the form must be sterile and mustbe fluid to the extent that easy syringability exists. It must be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms such as bacteria andfungi. The carrier can be a solvent or dispersion medium containing, forexample, sterile water, ethanol, polyol (for example, glycerol,propylene glycol and liquid polyethylene glycol and the like), suitablemixtures thereof, and vegetable oils. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of adispersion, and by the use of surfactants. The preventions of the actionof microorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thermerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminium monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activematerial in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredient into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and the freeze-dryingtechnique which yield a powder of the active ingredient plus anyadditional desired ingredient from previously sterile-filtered solutionthereof.

When IL-4 is suitably protected as described above, the active compoundmay be orally administered, for example, with an inert diluent or withan edible carrier, or it may be enclosed in hard or soft shell gelatincapsule, or it may be compressed into tablets, or it may be incorporateddirectly with the food of the diet. For oral therapeutic administration,the active material may be incorporated with excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like.

The tablets, troches, pills, capsules and the like may also contain thefollowing: A binder such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, lactose or saccharin may be added or a flavouring agent such aspeppermint, oil of wintergreen, or cherry flavouring. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier. Various other materials may be present ascoatings or to otherwise modify the physical form of the dosage unit.For instance, tablets, pills, or capsules may be coated with shellac,sugar or both. A syrup or elixir may contain the active compound,sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavouring such as cherry or orange flavour. Ofcourse, any material used in preparing any dosage unit form should bepharamceutically pure and substantially non-toxic in the amountsemployed. In addition, the active material may be incorporated intosustained-release preparations and formulations.

As used herein, the terms "pharmaceutically acceptable carrier" and"excipient" include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like described above. The use of such carriers andexcipients is well known in the art, see for example, Remington'sPharmaceutical Science and U.S. Pharmacopeia (1984); Mack PublishingCompany, Easton, Pa.

The active material may also be administered in association with one ormore anti-thrombolytic agents selected from, for example, tPA,prourokinase, urokinase or streptokinase. Potentiation of fibrinolyticactivity may take place when IL-4 is administered with such agents.

The active material may also be administered in association with one ormore anticoagulant agents, such as heparin, warfarin, aspirin,anisindione, phenindone and bishydroxy coumarin; and/or one or morevasodilators such as nitriles (for example, amylnitrile, nitroglycerin,sodium nitrile, isosorbide dinitrate), papaverine, nicotinic acid andcyclandelate. Anticoagulant and vasodilatory agents may improve accessto thrombosis and other fibrin deposits thereby enhancing fibrindegradation.

The invention is also concerned in another aspect with thrombolyticcompositions which comprise IL-4 in association with one or morepharmaceutically acceptable carriers or excipients; and which optionallyinclude one or more anti-thrombolytic agents, and/or one or moreanticoagulant agents, and/or one or more vasodilators, as describedabove.

The action of IL-4 in causing fibrin degradation and preventing fibrinand thrombi formation is surprising and unexpected based on the priorart known to the applicant, and provides new therapies for diseasesassociated with fibrin formation.

The following Examples further illustrate the invention. It will, ofcourse, be understood that the invention is in no way restricted to thespecific embodiments described in these Examples.

EXAMPLE 1 Materials and Methods Monocyte Isolation and Culture

Monocytes were isolated from peripheral venous blood by countercurrentcentrifugal elutriation as described by Hart et al. (1988) J. Immunol.141: 1516. Cell fractions containing >95% monocytes, identified bymorphological criteria, by non-specific esterase staining and by theirphagocytic properties were pooled and cultured for 18 h as previouslydetailed (0.8-1.0×10⁶ in 1 ml α-Modified Eagle's Medium containing 1%fetal calf serum). To terminate the cultures, the supernatants werecentrifuged to remove non-adherent cells; after twice washing with PBS,the adherent and non-adherent cells were pooled and lysed with 0.2%Triton X-100 in PBS.

Assay for PA Activity

Monocyte supernatants or lysates (50 μl) and human plasminogen (0.8 μg),dissolved in 100 μl 0.1M Tris-HCl, pH 8.1, were added to 0.28 cm² wellspreviously coated with ¹²⁵ I-fibrin, according to the methods ofHamilton (1981) J. Immunol. 126: 851. After 2-3 h, soluble ¹²⁵ I-fibrindegradation products were measured. PA activity was expressed accordingto the activity of u-PA standards (Leo Pharmaceutical Products,Denmark). Monocyte-derived plasminogen-independent fibrinolytic activitywas always <5% of the plasminogen-dependent activity.

Antibody Analysis

IgGs (immunoglobulins) were isolated from rabbit antisera to human u-PAand to human t-PA (Lots 100 and 153, respectively provided by Dr. W-D.Schleuning, CHUV, Laus anne, Switzerland) by standard methods usingProtein A Sepharose CL-4B (Pharmacia, Uppsala, Sweden). The mousemyeloma IgG, HOPCY (Dr. A. Burgess, Ludwig Institute for CancerResearch, Melbourne, Australia), was used as an irrelevant antibody.Culture supernatants, cell lysates or PA standards (u-PA) as above, t-PAas a culture supernatant rom the MM138 melanoma cell line (Dr. R.Whitehead, Ludwig Institute for Cancer Research, Melbourne), 0.2 IU/ml)were incubated with IgGs (1 μg/ml final concentration) for 1 h at 37° C.prior to assay of residual PA activity. In the immunoprecipitationexperiments, Protein A Sepharose CL-4B was added, as well as TritonX-100 to a final concentration of 0.25%. After further incubation for 30min. at room temperature with periodical mixing, the pellets were washedas previously described in Vassalli, J. D., et al. (1984) J. Exp. Med.159: 1643, except that SDS was not included in the wash buffers.

SDS-Casein Zymography

SDS-PAGE zymography was carried out according to the methods of Roche etal. (1983) B.B.A. 745: 82. The resolving gel (10%) contained casein (2mg/ml, Sigma) and human plasminogen (6 μg/ml) and waspre-electrophoresed at 15 mA for 1 h at room temperature. Samples (20μl), equilibrated in 0.0625M Tris-HCl, pH 6.8, 1.25% SDS, 10% glycerol,were incubated at 70° C. for 10 min before loading onto the stacking gel(4%), followed by electrophoresis at 12 mA for 2 h using the buffersystem of Laemmli, Nature 227: 680 (1970). After electrophoresis, gelswere washed for 1 h in 2.5% Triton X-100, then rinsed and incubated in0.1M Tris, pH 8.0, for 18 h at 37° C. Gels were stained by immersion in0.25% coomassie blue R-250 in 50% methanol/7% acetic acid for 60 min,then destained for 2 h in 30% methanol/10% acetic acid.

Detection of t-PA.mRNA

Total monocyte RNA was prepared according to the methods of Chirgwin etal. (1979) Biochemistry 18: 5292) and fractionated (5 μg/lane) on aformaldehyde-containing 1% agarose gel prior to transfer to GenescreenPlus nylon membrane (Dupont, Boston, Mass.). The filter was hybridizedovernight at 60° C. in a standard hybridization buffer containing >2×10⁶cpm/ml of ³² P-labelled t-PA.cRNA. The cRNA probe was prepared from aplasmid containing the 2.3 kb Bgl II fragment from pPAll 4B (Fisher, R.et al. (1985) J. Biol. Chem. 260: 11223), cloned into the vector pGEM-4blue (Promega, Madison, Mass.) and linearized with XbaI prior toinitiation of transcription with T7 RNA polymerase (New England Biolabs,Beverly, Mass.). After hybridizaticn, the filter was washed three timeswith 2×SSC prior to treatment with 1 μg/ml RNase A (Boehringer-Mannheim,West Germany) for 20 min at 37° C.

Measurement of Procoagulant Activity

Monocyte procoagulant activity is measured by the ability of the cellsto shorten the partial thromboplastin (clotting) time of platelet poorplasma. The human monocytes (4×10⁶ cells/ml) are activated bylipopolysaccharide or a supernatant from stimulated lymphocytes for 18 hat 37° C. to generate procoagulant activity on their surface. Thisactivity of frozen-thawed and sonicated cell samples was tested usingcitrate-treated platelet Poor human plasma in a prothrombin assay.Briefly, the clotting assay was performed by incubating the cellsuspension 0.8×10⁵ cells/0.1 ml) with 0.1 ml plasma for 1 min. at 37° C.Then CaCl₂ (30 mM; 0.1 ml) was added and the clotting time determinedmanually. Results are cited as mU of activity/10⁶ cells. A standardcurve of the clotting time of human plasma with decreasing dilutions ofrabbit brain thromboplastin was used to calculate mU.

EXAMPLE 2 t-PA Production by IL-4 Stimulated Human Monocytes

Human monocytes prepared according to Example 1 (1×10⁶ /ml) wereincubated with 0.25 U/ml human recombinant IL-4 (Genzyme).

Plasminogen activator (PA) activity (determined by plasminogen-dependentfibrinolytic activity as described in Example 1) was detected inmonocyte lysates after exposure to IL-4 for 2 h, and was secreted intothe culture supernatants by 3 h, with maximal production of PA occurring6 h after IL-4 stimulation.

All of the PA activity detected in the monocyte culture supernatants wasblocked by anti-tPA IgG, but not by anti-urokinase IgG. By SDS-caseinzymography, a technique which determines the apparent molecular weightof PA, a 70 kD band migrating in a manner characteristic of the t-PAstandard was found. Accordingly, IL-4 stimulates human monocytes toproduce t-PA.

Northern analysis showed the presence of t-PA.mRNA in monocytes treatedwith IL-4. t-PA mRNA was not detected in unstimulated monocytes.

EXAMPLE 3 Production of PA Activity by Endothelial Cells Stimulated withIL-4

Bovine aortic endothelial cells (10⁵ cells) were incubated in RPMI/10%FCS for 48 h, washed in isotonic saline, and then cultured in α-MEM (1ml) in the presence or absence of 2.5 units/ml purified human IL-4(Genzyme). After 24 h, conditioned medium was collected and assayed forPA activity, as a plasminogen-dependent fibrinolytic activity. PAactivity was detected in those cells stimulated with IL-4. Results areas follows:

    ______________________________________                                                           PA Activity                                                                   Iu/ml                                                      ______________________________________                                        Bovine endothelial cells                                                                         0.09 ± 0.01                                             (Control)                                                                     Bovine endothelial cells                                                                         0.32 ± 0.01                                             stimulated with 2.5 units/ml                                                  ______________________________________                                    

IL-4SDS-casein zymography identified urokinase type PA activity and t-PAactivity with the increase being in the urokinase type.

EXAMPLE 4 Inhibition of Procoagulant Activity of Human Monocytes by IL-4

Human monocytes (4×10⁶ cells), prepared according to Example 1, wereincubated with 100 ng/ml lipopolysaccharide (LPS), with humanrecombinant IL-4 (Schering Plough), or with LPS together with varyingconcentrations of IL-4.

Procoagulant activity (PCA) (determined by the shortening of theclotting time of citrated plasma as described in Example 1) was measuredin monocyte lysates after exposure to the reagents for 18 h at roomtemperature.

    ______________________________________                                                           PCA                                                                           MU/10.sup.6 cells                                                             Experiment 1                                                                             Experiment 2                                    ______________________________________                                        Human monocytes (control)                                                                        N.D.       N.D.                                            Human monocytes treated with                                                                     N.D.       N.D                                             2.5 units/ml IL-4                                                             Human monocytes treated with:                                                 LPS (100ng/ml)     20.9 ± 0.8                                                                            19.4 ± 0.5                                   LPS (100ng/ml) + IL-4 (2.5 U/ml)                                                                  4.6 ± 0.1                                                                            1.4 ± 0.1                                    LPS (100ng/ml) + 0.5                                                                              4.2 ± 0.2                                                                            5.1 ± 0.2                                    LPS (100ng/ml) + 0.1 U/ml)                                                                       13.9 ± 0.4                                                                            8.5 ± 0.7                                    ______________________________________                                         N.D.=not detected.                                                       

The claims defining the invention are as follows:

We claim:
 1. A method for degrading fibrin deposits and preventing theformation of such deposits associated with pathological conditions orwhich may lead to such conditions, which comprises administering to asubject in need of such treatment a therapeutically effective amount ofIL-4 or a derivative thereof possessing IL-4 activity, optionally inassociation with one or more pharmaceutically acceptable carriers orexcipients.
 2. A method according to claim 1, wherein the pathologicalconditions treated are selected from deep vein thrombosis, pulmonaryembolism, renal disease, hypertrophic keloid scars, coronary infarction,metastasis, inflammation, disseminated intravascular coagulation,atherosclerosis, rheumatoid arthritis, glomerulonephritis, systemiclupus erythematosis, autoimmune neuropathies, granulomatous disease,parasitic infection, allograft rejection, and other conditionsassociated with fibrin deposition.
 3. A method according to claim 1,wherein the IL-4 or a derivative thereof is locally administered at ornear the site of fibrin deposition.
 4. A method according to claim 1,where the IL-4 or a derivative thereof is administered by intravenous,intramuscular, intranasal, intradermal, intraperitoneal, suppository ororal route.
 5. A method according to claim 1, where the therapeuticallyeffective amount of IL-4 or derivatives thereof is between about 0.1 μgto about 200 mg per Kg body weight of the subject to be treated.
 6. Amethod according to claim 1, where the IL-4 or a derivative thereof iscontinually infused into a subject for a predetermined period.
 7. Amethod according to claim 1, wherein the IL-4 or a derivative thereof isadministered in conjunction with one or more thrombolytic agents oractivators of thrombolytic agents selected from tPA, urokinase,streptokinase or prourokinase.
 8. A method according to claim 1, whereinthe IL-4 or a derivative thereof is administered in conjunction with oneor more anticoagulant agents such as heparin, warfarin, asprin,anisindione, phenindone and bishydroxy coumarin.
 9. A thrombolyticcomposition comprising IL-4 or a derivative thereof possessing IL-4activity in association with a pharmaceutically acceptable carrier orexcipient, and one or more thrombolytic agents or activators thereofselected from the group consisting of tPA, urokinase, prourokinase andstreptokinase.
 10. A thrombolytic composition according to claim 9,which additionally comprises one or more anticoagulant agents.
 11. Athrombolytic composition according to claim 10, wherein theanticoagulant agents are selected from the group consisting of heparin,warfarin, aspirin, anisindone, phenindone and bishydroxy coumarin.
 12. Athrmobolytic composition according to claims 9 or 10, which additionallycomprises one or more vasodilators.
 13. A composition according to claim12, wherein the vasodilators are selected from the group consisting ofnitriles, papaverine, nicotinic acid and cyclandelate.